CN108387745B - Application of CD4+ T lymphocyte characteristic protein in identification of latent tuberculosis infection and active tuberculosis - Google Patents

Abstract

The invention discloses application of CD4+ T lymphocyte characteristic protein in identifying active tuberculosis and latent tuberculosis infection. The invention provides application of a substance for detecting the expression quantity of integrin beta-3 and/or integrin-linked kinase in preparing products for distinguishing or identifying or assisting in distinguishing or identifying active tuberculosis patients and tuberculosis latent infectors. The invention discovers that integrin beta-3 and integrin-linked kinase proteins can distinguish LTBI patients from primarily treated active tuberculosis patients, and provides a new diagnosis standard for diagnosing the LTBI patients and primarily treated active tuberculosis patients.

Description

Application of CD4+ T lymphocyte characteristic protein in identification of latent tuberculosis infection and active tuberculosis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of CD4+ T lymphocyte characteristic protein in identification of active tuberculosis and latent tuberculosis infection.

Background

The world health organization in 2015 defined LTBI as a state in which there was no clinical manifestation of active tuberculosis, but the organism persisted in an immune response to MTB antigen stimulation in the "guideline for diagnosis and treatment of latent MTB (MTB) infection (1. ent TB infection). According to the world health organization, about 1/3 people worldwide infect mycobacterium tuberculosis, of which more than 95% represent latent tuberculosis infection, and of which 5-10% of LTBI infected persons eventually develop active tuberculosis, becoming the main source of infection. Many LTBI infectors are the key and difficult point of effective prevention and control of tuberculosis at present, so accurate diagnosis of latent infection of tubercle bacillus has very important significance for preventing tuberculosis.

Since the latent infected patients lack specific symptoms and there is no unified standard and method for diagnosing latent tuberculosis infection at present, it is difficult to rapidly detect the latent tuberculosis infection patients in healthy people. The methods used in clinical practice for LTBI screening are mainly: tuberculin Skin Test (TST), Interferon-gamma release assay (IGRA). TST is simple to perform, but cannot distinguish between MTB infection and bacillus Calmette-Guerin (BCG) vaccination, and is less sensitive in immunodeficient people. In areas with high prevalence of tuberculosis in China, TST can cause false positives due to widespread inoculation with BCG; and false negative easily appears in HIV infected patients, autoimmune diseases, the elderly, children and other people with low immunity. In addition, TST detection has the defects of time consumption (48-72 hours), requirement of return visit of a subject, subjective dependence of skin test operation and result explanation and the like. IGRA detects LTBI with greater specificity than TST and is not affected by BCG vaccination and non-tuberculous mycobacteria (NTM) infection. However, the current IGRA experiment only detects IFN-gamma molecule, has low sensitivity, is easily interfered by other factors of the organism, only can detect the infection of tubercle bacillus and cannot distinguish active tuberculosis infection from latent tuberculosis infection. Moreover, IGRA experiments require blood drawing to obtain lymphocytes, require professional operators and laboratories with corresponding conditions, and samples must be completed within 8 hours after collection, and thus, cannot completely replace TST at the present stage. In addition, the samples studied at the present stage are very small and there is no gold standard for diagnosing LTBI. Therefore, there is still further research on IGRA.

In addition, although there are also new markers such as: the emergence of interleukin-2 (IL-2), HSP16.3, interferon inducible protein 10(IP-10), and monocyte chemotactic protein 2(MCP-2), etc., but there is still insufficient clinical evidence.

The tubercle bacillus infection mainly causes cellular immunity, and T lymphocytes generate a series of cell factors of differentiation, proliferation, synthesis and secretion after being stimulated by antigens to generate specific cellular immunity. Among them, CD4⁺The T lymphocyte plays an important role in secreting lymphokines, activating and chemotactic macrophages, promoting the macrophages to completely kill mycobacterium tuberculosis and the like.

Integrins (integrins) are membrane surface adhesion molecules that mediate cell-to-cell, cell-to-extracellular matrix adhesion, and are involved in a series of important physiological and pathological processes such as cell signal transduction, growth, differentiation, apoptosis, extension and movement, inflammation, thrombosis, tumor metastasis, and wound healing. α IIb β 3 is the most studied molecule in the integrin family and is representative of the integrin family. Alpha IIb beta 3 is an integrin molecule which is most abundantly expressed on the surface of a platelet membrane and plays an important role in the hemostasis functions of platelet adhesion, aggregation, release and the like. Research shows that the signal of the integrin alpha IIb beta 3 can be divided into a signal from the inside of a membrane to the outside of the membrane and a signal from the outside of the membrane to the inside of the membrane, wherein the signal is induced by an agonist to cause platelet activation, change the conformation of the integrin alpha IIb beta 3 and enable the integrin alpha IIb beta 3 to be adhered to a ligand thereof; the latter is triggered by ligand binding to the integrin α IIb β 3 receptor to produce a series of platelet functions.

Integrin-linked kinase (ILK), an important regulatory protein in integrin signaling pathway, plays a crucial role in integrin signaling pathway activation. It is a serine/threonine protein kinase consisting of 452 amino acids and highly expressed in various tissues throughout the body, and protein domain analysis suggests that ILK contains an N-terminal Ankyrin Repeat (ARD) structure and a kinase-like structure with a C-terminal end bound to the cytoplasmic tail of integrin. It mediates signal transduction between cells and extracellular matrix (ECM) and even from cell to cell by binding with integrin subunits, thereby affecting the downstream transmission of extracellular signals and regulating cell growth, proliferation, differentiation, survival, migration, apoptosis, adhesion and invasion.

Disclosure of Invention

An object of the present invention is to provide the use of a substance for detecting the expression level of integrin beta-3 and/or integrin-linked kinase.

The substance for detecting the expression level of integrin beta-3 and/or integrin-linked kinase provided by the invention is applied to the preparation of products for distinguishing or identifying or assisting in distinguishing or identifying active tuberculosis patients and tuberculosis latent infectors.

Another object of the present invention is to provide the use of a substance for detecting the expression level of integrin alpha-IIb (integrin alpha-IIb), integrin beta-3 (integrin beta-3) and/or integrin-linked kinase.

The application of the substance for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase in preparing products for detecting or assisting in detecting whether the products are tuberculosis patients or not is provided;

or the substance for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase provided by the invention is applied to the preparation of products for detecting or assisting to detect whether the person is a tuberculosis latent infected person.

In the application, the substance for detecting the expression level of integrin alpha-IIb is 1) or 2) as follows:

1) reagents and/or instruments required for detecting the expression quantity of the integrin alpha-IIb by utilizing protein mass spectrometry;

2) reagents and/or instruments required for detecting the expression quantity of integrin alpha-IIb by using Western Blot;

and/or the substance for detecting the expression level of integrin beta-3 is the following 3) or 4):

3) reagents and/or instruments required for detecting the expression quantity of integrin beta-3 by utilizing protein mass spectrometry;

4) reagents and/or instruments required for detecting the integrin beta-3 expression quantity by using Western Blot;

and/or the substance for detecting the expression level of the integrin-linked kinase is the following 5) or 6):

5) a reagent and/or an instrument required for detecting the expression quantity of the integrins-linked kinase by utilizing protein mass spectrometry;

6) reagents and/or instruments required for detecting the expression level of integrin-linked kinase by using Western Blot.

In the application, the expression quantity of integrin alpha-IIb is CD4⁺Expression level in T lymphocytes;

and/or the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

and/or the expression level of integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

The 3 rd object of the present invention is to provide the use of a substance that distinguishes or assists in distinguishing between active tuberculosis patients and those with latent tuberculosis infection, with integrin beta-3 and/or integrin-linked kinase as a marker.

The invention provides an application of a substance which takes integrin beta-3 and/or integrin-linked kinase as a marker for distinguishing or assisting in distinguishing active tuberculosis patients and tuberculosis latent infectors in preparing products for distinguishing or assisting in distinguishing the active tuberculosis patients and the tuberculosis latent infectors;

or the invention provides the application of the substance which takes integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase as markers for detecting or assisting to detect whether the substance is a product for detecting or assisting to detect whether the substance is a patient with tuberculosis;

or the invention provides the application of the substance for detecting or assisting to detect whether the person is a latent tuberculosis infected person or not by taking integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase as markers in preparing products for detecting or assisting to detect whether the person is a latent tuberculosis infected person or not.

In the application, the substance for distinguishing or assisting to distinguish the active tuberculosis patients and the tuberculosis latent infected patients is a substance for detecting the expression quantity of integrin beta-3 and/or integrin-linked kinase;

or, the substance for detecting or assisting the detection of the pulmonary tuberculosis patient is a substance for detecting the expression level of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase;

or the substance for detecting or assisting the detection of the tuberculosis latent infected person is a substance for detecting the expression level of integrinalpha-IIb, integrinbeta-3 and/or integrinlinked kinase.

The 4 th object of the present invention is to provide the use of integrin beta-3 and/or integrin-linked kinase as a marker for distinguishing active tuberculosis patients from those with latent tuberculosis infection.

The invention provides application of integrin beta-3 and/or integrin-linked kinase as a marker for distinguishing active tuberculosis patients and tuberculosis latent infectors in distinguishing or assisting in distinguishing the active tuberculosis patients and the tuberculosis latent infectors;

or, the invention provides the application of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase as a marker for detecting or assisting to detect whether the patient is the tuberculosis patient or not in detecting or assisting to detect whether the patient is the tuberculosis patient or not;

or, the invention provides the application of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase as a marker for detecting or assisting to detect whether a patient is a tuberculosis patient or not in detecting or assisting to detect whether the patient is a tuberculosis latent infected patient or not.

The 5 th object of the present invention is to provide a substance that distinguishes or assists in distinguishing between active tuberculosis patients and those with latent tuberculosis infection.

The substance for distinguishing or assisting to distinguish the active tuberculosis patients and the tuberculosis latent infectors is a substance for detecting the expression quantity of integrin beta-3 and/or integrin-linked kinase;

or, the substance for detecting or assisting in detecting whether the substance is a substance for a patient with tuberculosis is a substance for detecting the expression level of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase;

or, the substance for detecting or assisting to detect whether the tuberculosis is a latent infected person is a substance for detecting the expression level of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase.

It is a 6 th object of the present invention to provide a method of distinguishing or aiding in distinguishing between active tuberculosis patients and those with latent tuberculosis infection.

The method provided by the invention comprises the steps of detecting the expression quantity of integrin beta-3 and/or integrin-linked kinase in a sample to be detected, and determining whether the sample to be detected is an active tuberculosis patient or a tuberculosis latent infected patient according to the expression quantity of the integrin beta-3 and/or integrin-linked kinase;

or, the method for detecting or detecting whether the patient is the patient with the pulmonary tuberculosis in an auxiliary way comprises the steps of detecting the expression level of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase in a sample to be detected, and determining whether the sample to be detected is the patient with the pulmonary tuberculosis according to the expression level of the integrin alpha-IIb, the integrin beta-3 and/or the integrin-linked kinase;

or, the method for detecting or detecting the tuberculosis latent infector in an auxiliary way comprises the steps of detecting the expression level of integrin alpha-IIb, integrin beta-3 and/or integrin-linked kinase in a sample to be detected, and determining whether the sample to be detected is the tuberculosis latent infector not according to the expression level of the integrin alpha-IIb, the integrin beta-3 and/or the integrin-linked kinase.

In the method, the sample to be detected is a mycobacterium tuberculosis infected person;

and/or the expression level of integrin alpha-IIb is CD4⁺Expression level in T lymphocytes;

and/or the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

and/or the expression level of integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

Experiments prove that the integrin alpha-IIb, the integrin beta-3 and the integrin-linked kinase protein can be used for distinguishing healthy people from LTBI patients or distinguishing healthy people from primarily treated active tuberculosis patients, more importantly, the integrin beta-3 and the integrin-linked kinase protein can be used for distinguishing LTBI patients from primarily treated active tuberculosis patients, and therefore a new diagnosis standard is provided for diagnosing the LTBI patients and primarily treated active tuberculosis patients.

Drawings

FIG. 1 is a standard curve for BCA protein quantification.

FIG. 2 shows the result of Western-based verification of CD4+ T lymphocyte signature protein.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 differentiation of active tuberculosis from latent tuberculosis infection CD4⁺Discovery and application of T lymphocyte characteristic protein

The following experimental instruments were liquid chromatography-electrospray ionization tandem mass spectrometry (Q-active Thermo Finnigan), electrophoresis (Bio-rad, USA), MultiSkans plate reader (Thermo, USA).

First, CD4⁺Collection of T lymphocytes

Blood samples of 9 cases each were collected from patients who had been clinically diagnosed with LTBI, patients who had been clinically diagnosed for the first treatment of active tuberculosis (to prevent the drug from affecting the body), and healthy controls. All blood samples were drawn in the early morning on an empty stomach and 5.0ml of peripheral blood was collected using a disposable vacuum anticoagulation tube.

Peripheral Blood Mononuclear Cell (PBMC) cells were extracted with lymphocyte isolate (Lymphoprep, Stemcell) within 2 hours, followed by isolation of CD4⁺T lymphocyte (CD 4)⁺T Cell Isolation Kit, Miltenyi), flow cytometry extracted CD4 was detected⁺The purity of T lymphocyte is 94.71%.

Second, mass spectrometric detection of CD4⁺T lymphocyte signature proteins

1. Protein extraction

1) Pre-cooling mortar in-20 deg.C refrigerator, taking out frozen CD4 from-80 deg.C refrigerator⁺T lymphocyte cell samples.

2) The cell suspension is transferred into a precooled centrifugal tube without a heat source, and is added into 0.3ml of protein lysate for full oscillation, so that the cells are dispersed and broken to the maximum extent until the solution is transparent and uniform.

3) Add 0.1ml of protein lysate and shake together until no visible suspension is visible, transfer to a pre-chilled 1.5ml tube.

4) And (5) detecting whether the volume of each tube of the ground objects is consistent or not, and not finding that the ground objects are too few.

5) The ultrasonication was set to 2 seconds at intervals of 10 seconds for 5 times and the whole operation was carried out on ice.

6) The sonicated material was placed on ice for 20 minutes, centrifuged at 10,000g for 30 minutes at 4 ℃ and the supernatant was transferred to a new tube to obtain CD4 from 9 patients with LTBI⁺T lymphocyte protein, CD4 from 9 patients with primary treatment of active tuberculosis⁺T lymphocyte protein and CD4 derived from 9 healthy controls⁺T lymphocyte protein, stored in a refrigerator at-80 ℃.

The formula of the protein lysate comprises: 8.0M Urea 100mM Tris-HCl pH 8.010 mM DTT 1 Xprotease inhibitor (Roche).

2. Protein quantification

CD4 from 9 LTBI patients extracted from above 1⁺T lymphocyte protein, CD4 from 9 patients with primary treatment of active tuberculosis⁺T lymphocyte protein and CD4 derived from 9 healthy controls⁺The T lymphocyte protein is respectively taken as the protein to be detected to be quantitatively detected as follows:

1) the final total protein amounts were 0. mu.g, 2.5. mu.g, 5. mu.g, 10. mu.g, 15. mu.g, 20. mu.g and 25. mu.g, respectively, and two detection points were set for the same protein concentration calibration curve.

2) Protein solution was prepared according to the calculation and BCA was added in a calculated volume, with a total volume of 25. mu.l. At the same time, 1ul of sample is prepared and mixed with 25ul of water.

3) According to the following weight ratio of 50: 1, uniformly mixing the solution A and the solution B of the BCA reagent to generate a working solution, adding 200 mu l of the working solution into each tube, and uniformly mixing. Adding a detection enzyme label plate.

4) Incubate at 37 ℃ for 30 minutes, then allow to stand at room temperature for 5 minutes. The presence of a purple gradient was visually observed. If the gradient is not obvious, the next detection is terminated.

5) The detection was carried out by a microplate reader (BioTek Co., Ltd.) and the detection wavelength was set at 562 nm.

6) And generating a standard curve according to the detection result, if the correlation R is less than 0.90, the standard curve cannot be used for calculation, and carrying out the experiment again.

7) According to a standard curve (fig. 1, linear formula: Y0.05422X +0.14396, R2 0.9984) to derive the total amount of test protein and calculate the corresponding concentration.

Note that:

1. the preparation before the detection of the protein sample ensures that the sample is fully dissolved, has no suspended matters and turbidity, has no precipitate after standing, and uses a buffer substance compatible with the BCA reagent.

2. The total amount of the protein to be detected is adjusted within the detection interval according to the amount of the starting material of the sample.

The results are shown in Table 1, where sample numbers 1-3 are the pool of 9 normal 3 specimens, sample numbers 4-6 are the pool of 9 latent tuberculosis infected 3 specimens, and sample numbers 7-9 are the pool of 9 active tuberculosis 3 specimens.

Table 1 shows the sample concentration and total amount distribution measured by BCA protein quantitation method

3. Trypsin enzymolysis sample

(1) Putting 200 mu g of protein solution into a centrifuge tube, and fixing the system to 125 mu l by 8M urea solution after 2 quantification;

(2) adding 5 mul of 1M DTT solution prepared in situ into the system, mixing uniformly, and incubating for 1h at 37 ℃;

(3) adding 20 mul of 1M IAA solution prepared in situ into the system, uniformly mixing, keeping out of the sun, and reacting for 1h at room temperature (25 ℃);

(4) sucking all samples, adding the samples into a 10kD ultrafiltration tube, centrifuging at 12000rpm (no more than 14000g) for 20min, and discarding the solution at the bottom of the collection tube;

(5) add 100. mu.l urea solution (8M) to the ultrafiltration tube, centrifuge at 12000rpm for 10min, and repeat 2 times.

(6) Adding 2-4 μ g (with the mass ratio of 1:50-100) of trypsin (100U/μ g) into an ultrafiltration tube, reacting at 37 ℃ for 20h at 50 μ l; and centrifuging at 12000rpm for 20min the next day, centrifuging the peptide fragment solution after enzymolysis and digestion, and collecting the precipitate at the bottom of the tube to obtain an enzymolysis product.

4. Liquid phase fractionation and protein mass spectrometry detection

The enzymatic hydrolysate obtained in 3 above was collected and 10. mu.g of the product was subjected to LCMSMS analysis according to the quantitative results. Separation was carried out using a nano liter flow rate HPLC liquid phase system EASY-nLC 1000. The liquid phase A was an aqueous solution containing 2% (by volume) acetonitrile and 0.1% (by volume) formic acid (i.e., the composition was 2ml acetonitrile, 0.1ml formic acid, and the balance water, calculated on the basis of 100 ml), and the liquid phase B was an aqueous solution containing 84% (by volume) acetonitrile and 0.1% (by volume) formic acid (i.e., the composition was 84ml acetonitrile, 0.1ml formic acid, and the balance water, calculated on the basis of 100 ml). The column Thermo EASY column SC200150 μm × 100mm (RP-C18) was equilibrated with 100% of solution A. Samples were loaded onto Thermo EASY column SC001 taps 150 μm.times.20 mm (RP-C18) (Thermo) by an autosampler and separated by chromatography at a flow rate of 400 nl/min. The relevant liquid phase gradients are as follows: 0min-100min, the linear gradient of the B liquid is from 0% to 45% (volume percentage content, the same below); 100min-108min, the linear gradient of the liquid B is from 45% to 100%; 108min-120min, and maintaining the solution B at 100%. The enzymolysis product is separated by capillary high performance liquid chromatography and then is subjected to mass spectrometry by using a Q-exact mass spectrometer (Thermo Finnigan). Analysis duration: 120min, detection mode: positive ion, parent ion scan range: 300-1800m/z, mass-to-charge ratios of polypeptides and fragments of polypeptides were collected as follows: 10 fragment patterns (MS2scan, HCD) were acquired after each full scan (full scan). The resolution of MS 1at M/Z200 is 70000, and the resolution of MS2 at M/Z200 is 17500.

5. Identification by mass spectrometry

First, the primary mass spectrum quantitative information is extracted by using Trans-genomic Pipeline software (adopting software default parameters), and the quantification is carried out by using Profile analysis2.0 software (adopting software default parameters). Then, the ProteinScaphe 2.1 software is used for data retrieval and integration, and the details are as follows:

and screening and filtering the data by using Peptide FDR (full-automatic detection) of less than or equal to 0.05. Maxquant software (part of ProteinScaphe 2.1 software) was introduced for Label-free analysis. The main parameters are as follows:

Main search ppm：6Missed cleavage：2

MS/MS tolerance ppm：20De-Isotopic：TRUE

enzyme：Trypsin database：ipi.human.3.87.fasta

Fixed modification：Carbamidomethyl(C)

Variable modification：Oxidation(M)，Acetyl(Protein N-term)

Decoy database pattern：reverse

Lable free quantification(LFQ)：TRUE

LFQ min ratio count：2

Match between runs：2min

Peptide FDR：0.05

Protein FDR：0.05

3 different proteins with fold change (ratio) > 1.5 or <0.6 are screened, so as to obtain a characteristic spectrum for distinguishing the proteins of the patients. Wherein, the protein with the ratio more than 1.5 is the protein with up-regulated expression, the protein with the ratio less than 0.6 is the protein with down-regulated expression, and the ratio is the ratio of the average expression quantity of the corresponding protein of the A patient to the average expression quantity of the corresponding protein of the B patient.

The finally obtained protein characteristic spectrum for distinguishing the patients A and B contains 4 proteins with expression difference, and the proteins with difference in comparison between the patients A and B comprise proteins with up-regulated expression and proteins with down-regulated expression.

The results are shown in Table 2 below,

table 2 shows proteins that differ among 3 groups of patients

It can be seen that the proteins with differential expression in the LTBI group and the active tuberculosis group are upregulated proteins compared to the healthy control group: integrin alpha-IIb (integrin alpha-IIb, sequence 1), integrin beta-3 (integrin beta-3, sequence 2), integrin-linked kinase (integrin linked kinase, sequence 3); wherein, the integrin beta-3 and the integrin-linked kinase can also effectively distinguish LTBI patients from active tuberculosis patients, and the expression level is higher in the active tuberculosis patients (P < 0.0001).

The threshold value calculation formula is as follows: relative expression of LTBI group/relative expression of healthy group

Distinguishing threshold values of health and LTBI, wherein if the relative expression level of integrin alpha-IIb in the sample to be detected is more than 2.59, the relative expression level of integrin beta-3 is more than 3.70 and/or the relative expression level of integrin kinase is more than 1.58 (at least one of the expression levels is met), the sample to be detected is LTBI, and if the condition is not met, the sample to be detected is healthy;

the threshold value calculation formula is as follows: relative expression of pulmonary tuberculosis group/relative expression of healthy group

Distinguishing threshold values (including activity and LTBI) of healthy and tuberculosis patients, if the relative expression level of integrin alpha-IIb in the sample to be detected is more than 3.04, the relative expression level of integrin beta-3 is more than 5.50 and/or the relative expression level of integrin kinase is more than 2.42 (at least one of the expression levels is met), determining that the sample to be detected is the tuberculosis patient, and if the condition is not met, determining that the sample to be detected is healthy;

the threshold value calculation formula is as follows: relative expression of pulmonary tuberculosis group/relative expression of LTBI group

And (3) distinguishing the LTBI from the active tuberculosis patient, if the relative expression level of integrin beta-3 in the sample to be detected is more than 1.49 and/or the relative expression level of integrin-linked kinase is more than 1.54 (at least one of the expression levels is met), determining that the sample to be detected is the LTBI patient, and if the condition is not met, determining that the sample to be detected is the active tuberculosis patient.

Third, Western verification of CD4+ T lymphocyte characteristic protein

1. Reagent and consumable

2. Extraction of cell proteins

Precooling RIPA protein extraction reagent, and adding protease inhibitor (phosphatase inhibitor is required to be added simultaneously for phosphorylation protein). 0.1M PMSF stock solution was added before the start of protein extraction, PMSF final concentration 1 mM. Cell count, in number of cells 1X 10⁷To the above-mentioned one collected each group of CDs 4⁺The T lymphocytes are added with 1ml of lysate, the cells are fully suspended by blowing with a gun head, and after completion, the cells are incubated on ice for 20min, centrifuged at 4 ℃, and centrifuged at 13000rpm for 20 min. And (4) after centrifugation, taking the supernatant, subpackaging and storing to obtain the protein.

BCA method proteinQuantification, results from CD4 of 9 patients with LTBI⁺T lymphocyte protein, CD4 from 9 patients with primary treatment of active tuberculosis⁺T lymphocyte protein and CD4 derived from 9 healthy controls⁺The amounts of T lymphocyte proteins are shown in Table 1, respectively.

3. WB detection of target protein

According to the molecular weight of the target protein, 12% and 8% of separation gel is prepared, and the concentration of the concentrated gel is 5%. Loading amount of the protein sample to be detected: 10 ug/well. Electrophoresis conditions: the concentrated gel has constant pressure of 90V for about 20 min; the separation gel was kept at a constant pressure of 160V and the electrophoresis stop time was determined by prestained protein marker. Wet transfer method, transfer conditions: 300mA constant current, a NC membrane with the aperture of 0.45um and the membrane rotating time of 2 h. And dyeing the membrane by using a ponceau red dyeing reagent after the membrane transferring is finished, and observing the membrane transferring effect. And (3) sealing: the membrane was completely immersed in 3% BSA-TBST and shaken gently at room temperature for 30 min. Primary antibody incubation: the primary antibody was diluted with 3% BSA-TBST, incubated for 10min at room temperature, and left overnight at 4 ℃.

Table 4 names of the primary antibodies used

Numbering	First antibody name	Dilution ratio	Volume after dilution
				1	Mrp8	1:2000	4ml
2	COX5B	1:1000	4ml
				3	BCS1L	1:1000	4ml
4	CD7	1:2000	4ml

The next day the membranes were removed from 4 degrees and incubated at room temperature for 30 min. Washing the membrane: the membrane was washed 5 times with TBST, 3min each time. And (3) secondary antibody incubation: the secondary antibody was diluted with 5% skimmed milk powder-TBST, goat anti-rabbit IgG (H + L) HRP, and gently shaken at room temperature 1:10000 for 40 min. Washing the membrane: the membrane was washed 6 times with TBST, 3min each time. ECL is added on the membrane and reacts for 3-5min, and the film is exposed: 10s-5min (exposure time is adjusted according to different light intensities), developing for 2min, and fixing.

The results after film scanning were as follows:

official experiment of reference protein WB

Washing the membrane with striping Buffer at 37 deg.C for 30min (if the molecular weight difference between the target protein and the reference protein is above 10K, the step of washing the membrane with striping Buffer can be omitted).

Washing the membrane: the membrane was washed 3 times with deionized water.

Washing the membrane: washing membrane with TBST for 3 times, each for 3min

The membrane was completely immersed in 5% skimmed milk powder-TBST and gently shaken at room temperature for 30 min.

Incubation internal reference: GAPDH murine mab was added, the antibody diluted with 5% skim milk powder-TBST, 1: 20000 dilution and incubation at room temperature for 40 min.

Washing the membrane: the membrane was washed 5 times with TBST, 3min each time.

And (3) secondary antibody incubation: the secondary antibody was diluted with 5% skim milk powder-TBST, goat anti-mouse IgG (H + L) HRP, 1:10000, and gently shaken at room temperature for 40 min.

Washing the membrane: the membrane was washed 5 times with TBST, 3min each time.

ECL is added on the membrane and reacts for 3-5min, and the film is exposed: 10s-5min (exposure time is adjusted according to different light intensities), developing for 2min, and fixing.

Relative expression quantity is the gray value of target protein/GAPDH gray value

The results are shown in FIG. 2, and it can be seen that the relative expression levels of integrin alpha-IIb, integrin beta-3 and integrin-linked kinase can distinguish LTBI groups from healthy control groups; the relative expression levels of integrin beta-3 and integrin-linked kinase allow differentiation between LTBI patients and active tuberculosis patients.

Sequence listing

<110> Beijing thoracic Hospital affiliated to capital medical university

Application of <120> CD4+ T lymphocyte characteristic protein in identification of latent tuberculosis infection and active tuberculosis

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1039

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Arg Ala Leu Cys Pro Leu Gln Ala Leu Trp Leu Leu Glu Trp

1 5 10 15

Val Leu Leu Leu Leu Gly Pro Cys Ala Ala Pro Pro Ala Trp Ala Leu

20 25 30

Asn Leu Asp Pro Val Gln Leu Thr Phe Tyr Ala Gly Pro Asn Gly Ser

35 40 45

Gln Phe Gly Phe Ser Leu Asp Phe His Lys Asp Ser His Gly Arg Val

50 55 60

Ala Ile Val Val Gly Ala Pro Arg Thr Leu Gly Pro Ser Gln Glu Glu

65 70 75 80

Thr Gly Gly Val Phe Leu Cys Pro Trp Arg Ala Glu Gly Gly Gln Cys

85 90 95

Pro Ser Leu Leu Phe Asp Leu Arg Asp Glu Thr Arg Asn Val Gly Ser

100 105 110

Gln Thr Leu Gln Thr Phe Lys Ala Arg Gln Gly Leu Gly Ala Ser Val

115 120 125

Val Ser Trp Ser Asp Val Ile Val Ala Cys Ala Pro Trp Gln His Trp

130 135 140

Asn Val Leu Glu Lys Thr Glu Glu Ala Glu Lys Thr Pro Val Gly Ser

145 150 155 160

Cys Phe Leu Ala Gln Pro Glu Ser Gly Arg Arg Ala Glu Tyr Ser Pro

165 170 175

Cys Arg Gly Asn Thr Leu Ser Arg Ile Tyr Val Glu Asn Asp Phe Ser

180 185 190

Trp Asp Lys Arg Tyr Cys Glu Ala Gly Phe Ser Ser Val Val Thr Gln

195 200 205

Ala Gly Glu Leu Val Leu Gly Ala Pro Gly Gly Tyr Tyr Phe Leu Gly

210 215 220

Leu Leu Ala Gln Ala Pro Val Ala Asp Ile Phe Ser Ser Tyr Arg Pro

225 230 235 240

Gly Ile Leu Leu Trp His Val Ser Ser Gln Ser Leu Ser Phe Asp Ser

245 250 255

Ser Asn Pro Glu Tyr Phe Asp Gly Tyr Trp Gly Tyr Ser Val Ala Val

260 265 270

Gly Glu Phe Asp Gly Asp Leu Asn Thr Thr Glu Tyr Val Val Gly Ala

275 280 285

Pro Thr Trp Ser Trp Thr Leu Gly Ala Val Glu Ile Leu Asp Ser Tyr

290 295 300

Tyr Gln Arg Leu His Arg Leu Arg Gly Glu Gln Met Ala Ser Tyr Phe

305 310 315 320

Gly His Ser Val Ala Val Thr Asp Val Asn Gly Asp Gly Arg His Asp

325 330 335

Leu Leu Val Gly Ala Pro Leu Tyr Met Glu Ser Arg Ala Asp Arg Lys

340 345 350

Leu Ala Glu Val Gly Arg Val Tyr Leu Phe Leu Gln Pro Arg Gly Pro

355 360 365

His Ala Leu Gly Ala Pro Ser Leu Leu Leu Thr Gly Thr Gln Leu Tyr

370 375 380

Gly Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Arg Asp

385 390 395 400

Gly Tyr Asn Asp Ile Ala Val Ala Ala Pro Tyr Gly Gly Pro Ser Gly

405 410 415

Arg Gly Gln Val Leu Val Phe Leu Gly Gln Ser Glu Gly Leu Arg Ser

420 425 430

Arg Pro Ser Gln Val Leu Asp Ser Pro Phe Pro Thr Gly Ser Ala Phe

435 440 445

Gly Phe Ser Leu Arg Gly Ala Val Asp Ile Asp Asp Asn Gly Tyr Pro

450 455 460

Asp Leu Ile Val Gly Ala Tyr Gly Ala Asn Gln Val Ala Val Tyr Arg

465 470 475 480

Ala Gln Pro Val Val Lys Ala Ser Val Gln Leu Leu Val Gln Asp Ser

485 490 495

Leu Asn Pro Ala Val Lys Ser Cys Val Leu Pro Gln Thr Lys Thr Pro

500 505 510

Val Ser Cys Phe Asn Ile Gln Met Cys Val Gly Ala Thr Gly His Asn

515 520 525

Ile Pro Gln Lys Leu Ser Leu Asn Ala Glu Leu Gln Leu Asp Arg Gln

530 535 540

Lys Pro Arg Gln Gly Arg Arg Val Leu Leu Leu Gly Ser Gln Gln Ala

545 550 555 560

Gly Thr Thr Leu Asn Leu Asp Leu Gly Gly Lys His Ser Pro Ile Cys

565 570 575

His Thr Thr Met Ala Phe Leu Arg Asp Glu Ala Asp Phe Arg Asp Lys

580 585 590

Leu Ser Pro Ile Val Leu Ser Leu Asn Val Ser Leu Pro Pro Thr Glu

595 600 605

Ala Gly Met Ala Pro Ala Val Val Leu His Gly Asp Thr His Val Gln

610 615 620

Glu Gln Thr Arg Ile Val Leu Asp Cys Gly Glu Asp Asp Val Cys Val

625 630 635 640

Pro Gln Leu Gln Leu Thr Ala Ser Val Thr Gly Ser Pro Leu Leu Val

645 650 655

Gly Ala Asp Asn Val Leu Glu Leu Gln Met Asp Ala Ala Asn Glu Gly

660 665 670

Glu Gly Ala Tyr Glu Ala Glu Leu Ala Val His Leu Pro Gln Gly Ala

675 680 685

His Tyr Met Arg Ala Leu Ser Asn Val Glu Gly Phe Glu Arg Leu Ile

690 695 700

Cys Asn Gln Lys Lys Glu Asn Glu Thr Arg Val Val Leu Cys Glu Leu

705 710 715 720

Gly Asn Pro Met Lys Lys Asn Ala Gln Ile Gly Ile Ala Met Leu Val

725 730 735

Ser Val Gly Asn Leu Glu Glu Ala Gly Glu Ser Val Ser Phe Gln Leu

740 745 750

Gln Ile Arg Ser Lys Asn Ser Gln Asn Pro Asn Ser Lys Ile Val Leu

755 760 765

Leu Asp Val Pro Val Arg Ala Glu Ala Gln Val Glu Leu Arg Gly Asn

770 775 780

Ser Phe Pro Ala Ser Leu Val Val Ala Ala Glu Glu Gly Glu Arg Glu

785 790 795 800

Gln Asn Ser Leu Asp Ser Trp Gly Pro Lys Val Glu His Thr Tyr Glu

805 810 815

Leu His Asn Asn Gly Pro Gly Thr Val Asn Gly Leu His Leu Ser Ile

820 825 830

His Leu Pro Gly Gln Ser Gln Pro Ser Asp Leu Leu Tyr Ile Leu Asp

835 840 845

Ile Gln Pro Gln Gly Gly Leu Gln Cys Phe Pro Gln Pro Pro Val Asn

850 855 860

Pro Leu Lys Val Asp Trp Gly Leu Pro Ile Pro Ser Pro Ser Pro Ile

865 870 875 880

His Pro Ala His His Lys Arg Asp Arg Arg Gln Ile Phe Leu Pro Glu

885 890 895

Pro Glu Gln Pro Ser Arg Leu Gln Asp Pro Val Leu Val Ser Cys Asp

900 905 910

Ser Ala Pro Cys Thr Val Val Gln Cys Asp Leu Gln Glu Met Ala Arg

915 920 925

Gly Gln Arg Ala Met Val Thr Val Leu Ala Phe Leu Trp Leu Pro Ser

930 935 940

Leu Tyr Gln Arg Pro Leu Asp Gln Phe Val Leu Gln Ser His Ala Trp

945 950 955 960

Phe Asn Val Ser Ser Leu Pro Tyr Ala Val Pro Pro Leu Ser Leu Pro

965 970 975

Arg Gly Glu Ala Gln Val Trp Thr Gln Leu Leu Arg Ala Leu Glu Glu

980 985 990

Arg Ala Ile Pro Ile Trp Trp Val Leu Val Gly Val Leu Gly Gly Leu

995 1000 1005

Leu Leu Leu Thr Ile Leu Val Leu Ala Met Trp Lys Val Gly Phe

1010 1015 1020

Phe Lys Arg Asn Arg Pro Pro Leu Glu Glu Asp Asp Glu Glu Gly

1025 1030 1035

Glu

<210> 2

<211> 788

<212> PRT

<213> Artificial sequence

<400> 2

Met Arg Ala Arg Pro Arg Pro Arg Pro Leu Trp Ala Thr Val Leu Ala

1 5 10 15

Leu Gly Ala Leu Ala Gly Val Gly Val Gly Gly Pro Asn Ile Cys Thr

20 25 30

Thr Arg Gly Val Ser Ser Cys Gln Gln Cys Leu Ala Val Ser Pro Met

35 40 45

Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro Leu Gly Ser Pro Arg Cys

50 55 60

Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn Cys Ala Pro Glu Ser Ile

65 70 75 80

Glu Phe Pro Val Ser Glu Ala Arg Val Leu Glu Asp Arg Pro Leu Ser

85 90 95

Asp Lys Gly Ser Gly Asp Ser Ser Gln Val Thr Gln Val Ser Pro Gln

100 105 110

Arg Ile Ala Leu Arg Leu Arg Pro Asp Asp Ser Lys Asn Phe Ser Ile

115 120 125

Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp Ile Tyr Tyr Leu Met

130 135 140

Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Trp Ser Ile Gln Asn Leu

145 150 155 160

Gly Thr Lys Leu Ala Thr Gln Met Arg Lys Leu Thr Ser Asn Leu Arg

165 170 175

Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Val Ser Pro Tyr Met Tyr

180 185 190

Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro Cys Tyr Asp Met Lys Thr

195 200 205

Thr Cys Leu Pro Met Phe Gly Tyr Lys His Val Leu Thr Leu Thr Asp

210 215 220

Gln Val Thr Arg Phe Asn Glu Glu Val Lys Lys Gln Ser Val Ser Arg

225 230 235 240

Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Thr

245 250 255

Val Cys Asp Glu Lys Ile Gly Trp Arg Asn Asp Ala Ser His Leu Leu

260 265 270

Val Phe Thr Thr Asp Ala Lys Thr His Ile Ala Leu Asp Gly Arg Leu

275 280 285

Ala Gly Ile Val Gln Pro Asn Asp Gly Gln Cys His Val Gly Ser Asp

290 295 300

Asn His Tyr Ser Ala Ser Thr Thr Met Asp Tyr Pro Ser Leu Gly Leu

305 310 315 320

Met Thr Glu Lys Leu Ser Gln Lys Asn Ile Asn Leu Ile Phe Ala Val

325 330 335

Thr Glu Asn Val Val Asn Leu Tyr Gln Asn Tyr Ser Glu Leu Ile Pro

340 345 350

Gly Thr Thr Val Gly Val Leu Ser Met Asp Ser Ser Asn Val Leu Gln

355 360 365

Leu Ile Val Asp Ala Tyr Gly Lys Ile Arg Ser Lys Val Glu Leu Glu

370 375 380

Val Arg Asp Leu Pro Glu Glu Leu Ser Leu Ser Phe Asn Ala Thr Cys

385 390 395 400

Leu Asn Asn Glu Val Ile Pro Gly Leu Lys Ser Cys Met Gly Leu Lys

405 410 415

Ile Gly Asp Thr Val Ser Phe Ser Ile Glu Ala Lys Val Arg Gly Cys

420 425 430

Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile Lys Pro Val Gly Phe Lys

435 440 445

Asp Ser Leu Ile Val Gln Val Thr Phe Asp Cys Asp Cys Ala Cys Gln

450 455 460

Ala Gln Ala Glu Pro Asn Ser His Arg Cys Asn Asn Gly Asn Gly Thr

465 470 475 480

Phe Glu Cys Gly Val Cys Arg Cys Gly Pro Gly Trp Leu Gly Ser Gln

485 490 495

Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro Ser Gln Gln Asp Glu Cys

500 505 510

Ser Pro Arg Glu Gly Gln Pro Val Cys Ser Gln Arg Gly Glu Cys Leu

515 520 525

Cys Gly Gln Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly

530 535 540

Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu

545 550 555 560

Met Cys Ser Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp

565 570 575

Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr

580 585 590

Cys Met Ser Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu

595 600 605

Cys Gly Ser Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr Cys

610 615 620

Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys Thr Phe Lys Lys Glu Cys

625 630 635 640

Val Glu Cys Lys Lys Phe Asp Arg Gly Ala Leu His Asp Glu Asn Thr

645 650 655

Cys Asn Arg Tyr Cys Arg Asp Glu Ile Glu Ser Val Lys Glu Leu Lys

660 665 670

Asp Thr Gly Lys Asp Ala Val Asn Cys Thr Tyr Lys Asn Glu Asp Asp

675 680 685

Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser Gly Lys Ser Ile

690 695 700

Leu Tyr Val Val Glu Glu Pro Glu Cys Pro Lys Gly Pro Asp Ile Leu

705 710 715 720

Val Val Leu Leu Ser Val Met Gly Ala Ile Leu Leu Ile Gly Leu Ala

725 730 735

Ala Leu Leu Ile Trp Lys Leu Leu Ile Thr Ile His Asp Arg Lys Glu

740 745 750

Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg Ala Lys Trp Asp Thr Ala

755 760 765

Asn Asn Pro Leu Tyr Lys Glu Ala Thr Ser Thr Phe Thr Asn Ile Thr

770 775 780

Tyr Arg Gly Thr

785

<210> 3

<211> 452

<212> PRT

<213> Artificial sequence

<400> 3

Met Asp Asp Ile Phe Thr Gln Cys Arg Glu Gly Asn Ala Val Ala Val

1 5 10 15

Arg Leu Trp Leu Asp Asn Thr Glu Asn Asp Leu Asn Gln Gly Asp Asp

20 25 30

His Gly Phe Ser Pro Leu His Trp Ala Cys Arg Glu Gly Arg Ser Ala

35 40 45

Val Val Glu Met Leu Ile Met Arg Gly Ala Arg Ile Asn Val Met Asn

50 55 60

Arg Gly Asp Asp Thr Pro Leu His Leu Ala Ala Ser His Gly His Arg

65 70 75 80

Asp Ile Val Gln Lys Leu Leu Gln Tyr Lys Ala Asp Ile Asn Ala Val

85 90 95

Asn Glu His Gly Asn Val Pro Leu His Tyr Ala Cys Phe Trp Gly Gln

100 105 110

Asp Gln Val Ala Glu Asp Leu Val Ala Asn Gly Ala Leu Val Ser Ile

115 120 125

Cys Asn Lys Tyr Gly Glu Met Pro Val Asp Lys Ala Lys Ala Pro Leu

130 135 140

Arg Glu Leu Leu Arg Glu Arg Ala Glu Lys Met Gly Gln Asn Leu Asn

145 150 155 160

Arg Ile Pro Tyr Lys Asp Thr Phe Trp Lys Gly Thr Thr Arg Thr Arg

165 170 175

Pro Arg Asn Gly Thr Leu Asn Lys His Ser Gly Ile Asp Phe Lys Gln

180 185 190

Leu Asn Phe Leu Thr Lys Leu Asn Glu Asn His Ser Gly Glu Leu Trp

195 200 205

Lys Gly Arg Trp Gln Gly Asn Asp Ile Val Val Lys Val Leu Lys Val

210 215 220

Arg Asp Trp Ser Thr Arg Lys Ser Arg Asp Phe Asn Glu Glu Cys Pro

225 230 235 240

Arg Leu Arg Ile Phe Ser His Pro Asn Val Leu Pro Val Leu Gly Ala

245 250 255

Cys Gln Ser Pro Pro Ala Pro His Pro Thr Leu Ile Thr His Trp Met

260 265 270

Pro Tyr Gly Ser Leu Tyr Asn Val Leu His Glu Gly Thr Asn Phe Val

275 280 285

Val Asp Gln Ser Gln Ala Val Lys Phe Ala Leu Asp Met Ala Arg Gly

290 295 300

Met Ala Phe Leu His Thr Leu Glu Pro Leu Ile Pro Arg His Ala Leu

305 310 315 320

Asn Ser Arg Ser Val Met Ile Asp Glu Asp Met Thr Ala Arg Ile Ser

325 330 335

Met Ala Asp Val Lys Phe Ser Phe Gln Cys Pro Gly Arg Met Tyr Ala

340 345 350

Pro Ala Trp Val Ala Pro Glu Ala Leu Gln Lys Lys Pro Glu Asp Thr

355 360 365

Asn Arg Arg Ser Ala Asp Met Trp Ser Phe Ala Val Leu Leu Trp Glu

370 375 380

Leu Val Thr Arg Glu Val Pro Phe Ala Asp Leu Ser Asn Met Glu Ile

385 390 395 400

Gly Met Lys Val Ala Leu Glu Gly Leu Arg Pro Thr Ile Pro Pro Gly

405 410 415

Ile Ser Pro His Val Cys Lys Leu Met Lys Ile Cys Met Asn Glu Asp

420 425 430

Pro Ala Lys Arg Pro Lys Phe Asp Met Ile Val Pro Ile Leu Glu Lys

435 440 445

Met Gln Asp Lys

450

Claims (5)

1. The application of the substance for detecting the expression quantity of integrin beta-3 and integrin-linked kinase in preparing products for distinguishing or identifying the products for treating the patients with active tuberculosis and the patients with latent tuberculosis infection;

the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

the expression level of the integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

2. The application of the substance for detecting the expression quantity of integrin beta-3 and integrin-linked kinase in the preparation of products for assisting in distinguishing or identifying the initially treated active tuberculosis patients and tuberculosis latent infectors;

the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

the expression level of the integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

3. The application of the substances for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and integrin-linked kinase in preparing products for detecting who is primarily treating active tuberculosis patients;

or the application of the substances for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and integrin-linked kinase in the preparation of products for detecting the latent tuberculosis infectors;

the expression level of integrin alpha-IIb is CD4⁺Expression level in T lymphocytes;

the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

the expression level of the integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

4. The application of substances for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and integrin-linked kinase in preparing products for assisting in detecting who is a patient who primarily treats active tuberculosis;

or the application of the substances for detecting the expression quantity of integrin alpha-IIb, integrin beta-3 and integrin-linked kinase in the preparation of products for assisting in detecting the latent tuberculosis infectors;

the expression level of integrin alpha-IIb is CD4⁺Expression level in T lymphocytes;

the expression level of integrin beta-3 is CD4⁺Expression level in T lymphocytes;

the expression level of the integrin-linked kinase is CD4⁺Expression level in T lymphocytes.

5. Use according to any one of claims 1 to 4, characterized in that: the substance for detecting the expression level of integrin alpha-IIb is 1) or 2) as follows:

1) reagents and/or instruments required for detecting the expression quantity of the integrin alpha-IIb by utilizing protein mass spectrometry;

2) reagents and/or instruments required for detecting the expression quantity of integrin alpha-IIb by using Western Blot;

and/or the substance for detecting the expression level of integrin beta-3 is the following 3) or 4):

3) reagents and/or instruments required for detecting the expression quantity of integrin beta-3 by utilizing protein mass spectrometry;

4) reagents and/or instruments required for detecting the integrin beta-3 expression quantity by using Western Blot;

and/or the substance for detecting the expression level of the integrin-linked kinase is the following 5) or 6):

5) a reagent and/or an instrument required for detecting the expression quantity of the integrins-linked kinase by utilizing protein mass spectrometry;

6) reagents and/or instruments required for detecting the expression level of integrin-linked kinase by using Western Blot.

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